CUTANATM ChIC / CUT&RUN KitUser Manual Version 2.0

Copyright 2020 EpiCypher, Inc. All rights reserved.

This document may not be duplicated in part or in its entirety without prior written consent of EpiCypher, Inc.

EpiCypher® and SNAP-ChIP® are registered trademarks of EpiCypher. All other trademarks and trade names in this document are property of their respective corporations.

EpiCypher, Inc.PO Box 14453Durham, NC 27709

www.epicypher.comPh: 1-855-374-2461 F: 1-855-420-6111Email: info@epicypher.comTech Support: techsupport@epicypher.com

Upon receipt, store indicated components at 4°C, -20°C and room temperature (RT)

See p. 8-9 for storage instructions.

ChIC / CUT&RUN KitCUTANATM

Catalog No. 14-104848 ChIC / CUT&RUN Samples

Kit Version 2.0User Manual Version 2.0

Upon receipt, store indicated components at 4°C, -20°C and room temperature (RT)

See p. 8-9 for storage instructions.

Overview 5Background and Description 6Included in the Kit 8Materials Required but Not Supplied 10Notes Before Starting 11Experimental Protocol: Day 1 12 Section I: CUTANA Spike-in Controls & Buffer Prep (~1 hr) 12 Section II: Bead Activation (~30 min) 14 Section III: Binding Cells to Activated Beads (~30 min) 14 Section IV: Antibody Binding (~30 min + overnight) 15Experimental Protocol: Day 2 16 Section V: Binding of pAG-MNase (~30 min) 16 Section VI: Targeted Chromatin Digestion and Release (~3 hrs) 16 SectionVII:DNAPurification(~30min) 17Experimental Protocol: Library Preparation and NGS 19 Section VIII: NGS Library Preparation (~4 hrs) 19 Section IX: Analysis of Library Fragment Size (~1 hr) 20 Section X: Illumina Sequencing 21 Section XI: Data Analysis 21Quality Control Checks 22Appendix I: Experimental Normalization Using E. coli Spike-in DNA 32Appendix II: Protocol Variations 33 Sample Preparation: Adherent Cells 33 Sample Preparation: Cryopreservation 33 Sample Preparation: Tissues 33 Sample Preparation: Immune Cells 33 Sample Preparation: Nuclei 34 Sample Preparation: Cross-linking 36Appendix III: Frequently Asked Questions 38Appendix IV: Safety Datasheet 40References 47

Table of Contents

5

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a revolutionary genomic mapping strategy developed by the group of Dr. Steven Henikoff1. It builds on Chromatin ImmunoCleavage (ChIC) from Dr. Ulrich Laemmli2, wherein a fusion of Protein A to Micrococcal Nuclease (pA-MNase) is recruited to selectively cleave antibody-bound chromatin in situ3. In CUT&RUN, cells or nuclei are immobilized to a solid support, with pAG-MNase cleaved DNA fragments isolated from solution. The workflow is compatible with next-generation sequencing (NGS) to provide high quality genome-wide profiles of histone post-translational modifications (PTMs) and chromatin-associated proteins (e.g. TFs and chromatin remodelers; Figure 1).

Target-DNA complex diffuses out,

collect supernatant

Extract DNA & preparesequencing library

Immobilize & permeabilize cells

(or nuclei)

Add & activate pAG-MNase

to cleave target-DNA complex

Add antibody tohistone PTM or

chromatin-interacting protein

Next-generation sequencing and

data analysis

PCR amplify & clean up DNA to append barcoded

adapters for sequencing

Isolate & immobilize nuclei

Add & activatepAG-Tn5 to cleave

target-DNA complex& ligate sequencing

adapters

Add primary &secondary antibodyto histone PTM or

chromatin-interactingprotein

Next-generationsequencing and

data analysis

Overview

FIGURE 1

Overview of the CUTANA CUT&RUN protocol.

Historically, ChIP-seq is the leading approach for genome-wide mapping of histone PTMs and chromatin-associated proteins. In this approach, bulk chromatin is fragmented by sonication or enzymatic digestion. Target-specific fragments are then immunoprecipitated. Despite extensive optimization and stringent wash conditions, ChIP-seq requires large numbers of cells (typically 105 – 106 cells) and deep sequencing of both input chromatin and immunoprecipitated material (typically > 30 million reads each) to resolve signal from background.

ChIC and CUT&RUN have revolutionized the study of chromatin regulation by enabling targeted release of genomic fragments into solution. With this innovation, background is dramatically reduced, allowing high resolution genomic mapping for histone PTMs and chromatin-associated proteins using a small number of cells and only 3-8 million sequencing reads per sample (Figure 2). The streamlined workflow and cost savings make ChIC/CUT&RUN amenable to greater experimental through-put, allowing deeper and more rapid investigations to uncover epigenetic biology.

FIGURE 2

Representative genome browser tracks show CUTANA CUT&RUN results using 500,000 K562 cells. Clearpeakswiththeexpecteddistributionprofileareobservedusing3-8millionsequencingreadsper sample for a variety of epigenetic targets, including histone PTMs (H3K4me3, H3K27me3, H3K27ac), transcription factors (CTCF), epigenetic reader proteins (BRD4), epigenetic writer enzymes (MLL1), and chromatin remodelers (SMARCA4). Rabbit IgG antibody is shown as a negative control (top track).

[0-301]

[0-301]

[0-43]

[0-418]

[0-926]

[0-180]

[0-332]

[0-95]

IgG

H3K4me3

H3K27me3

H3K27ac

CTCF

BRD4

MLL

SMARCA4

Fig 2

TAL1 STIL CMPK1 LINC01389 FOXD2

Background and Description

7

The CUTANATM ChIC/CUT&RUN Kit contains sufficient materials for 48 samples and is designed for multi-channel sample pipetting in order to realize the increased experimental throughput advantage of CUT&RUN. The kit includes positive (H3K4me3) and negative (Rabbit IgG) control antibodies. A panel of four designer nucleosomes (H3K4me0, 1, 2, and 3 dNucsTM) are spiked-in to control samples to directly monitor experimental success and aid troubleshooting. Additionally, sheared E. coli DNA is added to samples after pAG-MNase cleavage to control for library preparation and enable experimental normalization. The kit is compatible with cells and nuclei, including cryopreserved and cross-linked samples (Figure 3 & Appendix II). Although it is recommended to start with 500,000 cells, comparable data can be generated using as few as 5,000 cells (Figure 4). The inclusion of rigorous controls as well as compatibility with diverse target types, sample inputs, and cell numbers make the CUTANA kit ideal for a variety of research applications.

Fig 4

[0-1415]

[0-1504]

[0-2279]

[0-760]

500k

50k

5k

0.5k

H3K4me3# cells:

DGCR8 TRMT2A RANBP1 ZDHHC8 LOC284865 RTN4R

[0-117]

[0-101]

[0-157]

[0-109]

H3K27me3

FIGURE 4

Representative genome browser tracks for H3K4me3 (low abundance target) and H3K27me3 (high abundance target) CUT&RUN experiments using decreasing amounts of K562 cells. At 5,000 cells, data quality is largely indistinguishable from standard conditions (500,000 cells).

FIGURE 3

Heatmaps showing CUT&RUN signal (red) and background (blue) of H3K4me3-enriched regions flankingannotatedtranscriptionstartsites(TSS,+/-2kb). Gene rows are aligned across the conditions, showing that the genome-wide enrichment pattern is preserved across sample types.

Fig 3

FreshCells

Freeze/Thawed

Cells

Cross-linkedCells Nuclei

18,7

93 g

enes

alig

ned

to T

SS

TSS-2kb +2kb

Included in the Kit

Store at 4°C upon receipt:

Store at -20°C upon receipt:

Item Catalog No. Notes before use

ConA Beads 21-1401 DO NOT FREEZE. Concanavalin A (ConA) beads are used for immobilizing cells or nuclei. ConA can cause immune cell activation. For immune cell studies, use nuclei as input (see Appendix II).

SA Beads 21-1402 DO NOT FREEZE. Streptavidin (SA)-conjugated magnetic beads are used to immobilize CUTANA H3K4 MetStat Spike-in Control dNucs.

Bead ActivationBuffer

21-1001 Use to prepare ConA beads prior to sample immobilization.

Pre-Wash Buffer 21-1002 Use to prepare Wash, Cell Permeabilization, and Antibody Buffers *FRESH* for each experiment.

Stop Buffer 21-1003 3X concentration: use to terminate pAG-MNase cleavage activity.

Item Catalog No. Notes before use

5% Digitonin 21-1004 Use to prepare Cell Permeabilization Buffer*FRESH* for each experiment. Final [digitonin] shoud be optimized for each sample type (see Quality Control Checks).

1 M Spermidine 21-1005 2,000X concentration. Use to prepare Wash Buffer *FRESH* for each experiment.

pAG-MNase 15-1016 Proteins A and G (pAG) are compatible with a variety of antibody isotypes.

H3K4me3 PositiveControl Antibody

13-0041k SMALL VOLUME: quick spin before use. Sufficient volume for 10 samples. Use as a positive control alongside targets of interest in every experiment.

Rabbit IgG NegativeControl Antibody

13-0042k SMALL VOLUME: quick spin before use. Sufficient volume for 10 samples. Use as a negative control alongside targets of interest in every experiment.

E. coli Spike-in DNA 18-1401 100 ng lyophilized E. coli DNA for data normalization. Before first use, quick spin and reconstitute in 200 μL DNase-free water (0.5 ng/μL).

CUTANA H3K4 Met-Stat Spike-in Controls

19-1006, 19-1321,19-1334,19-1316

SMALL VOLUME: quick spin before use. Unmodified, H3K4me1, H3K4me2 and H3K4me3 barcoded dNucs (60 nM). Use as a spike-in control with H3K4me3 and IgG control antibodies (see Quality Control Checks).

9

Item Catalog No. Notes before use

8-strip Tubes 10-0009k Compatible with multi-channel pipettors and Magnetic Separation Rack (EpiCypher Catalog No. 10-0008).

DNA CleanupColumns

10-0010 Use with the DNA Collection Tubes.

DNA CollectionTubes

10-0011 Use with the DNA Cleanup Columns.

0.5 M EDTA 21-1006 250X concentration. Use to prepare Antibody Buffer *FRESH* for each experiment.

100 mM Calcium Chloride

21-1007 When added to sample, this will activate chromatin tethered pAG-MNase to cleave DNA.

DNA Binding Buffer 21-1008 WARNING: Contains toxic ingredients (see Appendix IV).

DNA Wash Buffer 21-1009 Before first use, add 20 mL ≥ 95% ethanol.

DNA Elution Buffer 21-1010 Recover final CUT&RUN DNA in 6 – 20 μL depending on desired final concentration.

Store at room temperature (RT) upon receipt:

* NOTE: The kit contains sufficient materials for 48 CUT&RUN samples. Additional materials will need to be procured to fully perform the protocol.

MATERIALS:

• 1.5, 15 and 50 mL tubes

• Antibody to target of interest (user-dependent)

* NOTE: The kit contains H3K4me3 and IgG positive/negative control antibodies to optimize conditions and monitor experimental success. EpiCypher continues to conduct extensive investigations of antibody performance4. Contact techsupport@epicypher.com for recommendations.

• Protease inhibitor (e.g. cOmplete™, EDTA-free Protease Inhibitor Cocktail, Roche #11873580001)

• 0.4% Trypan blue (e.g. Invitrogen #T10282)

• Qubit™ 1X dsDNA HS Assay Kit (Invitrogen #Q33230)

• Library Preparation Kit (e.g. NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, New England Biolabs #E7645S)

• AMPure® XP magnetic beads (Beckman Coulter #A63880)

EQUIPMENT:

• 8-strip PCR Tube Magnetic Separation Rack (EpiCypher #10-0008)

• 1.5 mL Tube Magnetic Separation Rack (EpiCypher #10-0012)

• Qubit™ 4 Fluorometer (Invitrogen #Q33226)

• Capillary electrophoresis machine to assess fragment size distribution after library preparation (e.g. Agilent 2100 Bioanalyzer® #G2939A and Agilent High Sensitivity DNA Analysis Kit #5067-4626)

• 8-channel multi-channel pipettor (e.g. VWR #76169-250)

• Multi-channel reagent reservoir (e.g. Thermo Fisher Scientific #14-387-072)

• Tube Nutator for incubation steps (e.g. VWR #82007-202)

• Vortex (e.g. Vortex-Genie®, Scientific Industries #SI-0236)

Materials Required but Not Supplied

11

Notes Before Starting

This kit has been validated for genomic profiling of:

• Histone PTMs (e.g. lysine methylation, lysine acetylation, lysine ubiquitylation, etc.)• Transcription factors (e.g. CTCF, FOXA1, GATA3)• Chromatin remodeling ATPases (e.g. SMARCA4-SWI/SNF, SMARCA1/ISWI,

INO80, CHD1, CHD3)• Chromatin writers & readers (e.g. MLL1, BRD4)

1. Always include control conditions (e.g. positive and negative control antibodies with CUTANA Spike-in Control dNucs; see Quality Control Checks) to confirm experimental success and guide troubleshooting. This is especially critical when evaluating CUT&RUN using previously untested targets and/or antibodies.

2. The Experimental Protocol was optimized using 500,000 human K562 cells per sample but is compatible with as few as 5,000 cells.

3. The Experimental Protocol describes sample preparation using fresh, non- adherent cells. For adherent cells, do NOT use trypsin. For protocol variations, including adaptations for adherent cells, nuclei, cryopreserved samples, and crosslinked material, see Appendix II.

4. Take caution throughout the experiment to avoid ConA beads sticking to the sides and caps of tubes. They dry out easily, which can result in sample loss.

5. Although protocols with shortened incubation times have been published3, such changes can adversely impact yield and reproducibility; therefore they are not recommended.

6. To avoid digitonin precipitation and cell lysis, use the minimal concentration of digitonin that is needed to achieve efficient cell permeabilization. This should be empirically determined for different samples before proceeding with the full CUT&RUN experiment (see Quality Control Checks).

7. Paired-end (PE) sequencing (min 50 nucleotides) is recommended to accurately align reads to the CUTANA Spike-in Control dNucs. Further, PE reads provide information on (and enable bioinformatic filtering of) DNA insert fragment sizes and aids DNA footprinting (e.g. for transcription factor binding studies).

8. In CUT&RUN, 3-8 million reads provides adequate coverage for most targets.

* NOTE: Gather all reagents stored at 4ºC and -20ºC needed for Day 1 (ConA Beads, SA Beads, Bead Activation Buffer, Pre-Wash Buffer, Digitonin, Spermidine, H3K4me3 / IgG control antibodies, CUTANA Spike-in Controls). Place on ice to thaw or equilibrate.

SECTION I: CUTANA SPIKE-IN CONTROLS & BUFFER PREP (~1 HR)

Experimental Protocol: Day 1

A. Prepare immobilized CUTANA H3K4 MetStat Spike-in dNuc Controls by first thoroughly mixing SA Beads by pipetting. Transfer 4 µL into a 1.5 mL tube.

B. Add 150 µL Pre-Wash Buffer to beads and pipette mix. Place on magnet and remove supernatant (sup) by pipetting.

C. Remove from magnet and resuspend beads in 210 µL Pre-Wash Buffer. Aliquot 50 µL bead slurry into 4 x 1.5 mL tubes.

D. Add 1 μL CUTANA Spike-in Control Unmodifed dNuc to a 1.5 mL tube. Pipette mix. Repeat for remaining dNucs (H3K4me1, H3K4me2 and H3K4me3), adding each into a separate tube. Pipette mix and incubate 30 min at RT on nutator.

E. During incubation, make CUT&RUN buffers fresh the day of use (see workflow illustrated in Figure 5 and volume scaling table, p. 13). Add 1.8 mL of Pre-Wash Buffer per sample to a single conical tube labeled “Wash Buffer”.

FIGURE 5

Schematic of buffers to be prepared fresh the day of use. All volumes are per sample and include overfilltoaccountforpipettingerror.Scaleupvolumesaccordingtothetotalnumberofsamples.

13

F. Dissolve 1 protease inhibitor tablet (Roche) in 2 mL water (25X stock). Add 72 μL per sample to the Wash Buffer. Store remaining 25X stock for 12 weeks at -20ºC.

G. Dilute 1 M Spermidine 1:2,000 in the Wash Buffer. Store final buffer at RT.

H. Transfer 1.4 mL of Wash Buffer per sample into a new conical tube labeled “Cell Permeabilization Buffer”. Add 5% Digitonin (1:500 dilution)*.

* NOTE: A 500X dilution (0.01% final digitonin) is optimal to permeabilize K562, MCF7, and A549 cells. For other cell types, see Quality Control Checks for detailed instructions regarding optimization of permeabilization conditions.

I. Transfer 100 μL per sample of Cell Permeabilization Buffer into a new tube labeled “Antibody Buffer”. Add 0.5 M EDTA (1:250 dilution). Store final buffer on ice.

J. Store the remaining Cell Permeabilization Buffer at 4°C overnight (for Day 2 use).

K. Finish preparing the CUTANA H3K4 MetStat Spike-in Control dNucs. Quick spin tubes in microfuge to collect samples. Place on magnet and remove sup.

L. Remove tubes from magnet. Add 150 µL Pre-Wash Buffer to resuspend beads.

M. Place back on magnet and remove sup. Remove from magnet and resuspend beads in 5 µL Pre-Wash Buffer. Combine dNucs into a single tube (20 µL total).

N. Place back on magnet and remove sup. Remove from magnet and resuspend in 8 µL Antibody Buffer.

O. Place dNucs on ice for later use (Section IV). Use as spike-in controls with the H3K4me3 / IgG control antibodies. Use FRESH the same day of preparation.

Buf

fer

Sam

ple

Sca

ling

Cal

cula

tion

s:

NUMBER OF SAMPLES 1X 8X 16X

Wash Buffer - store at RT for use on Day 1

Pre-Wash Buffer 1.8 mL 14.4 mL 28.8 mL

25X Protease Inhibitor 72 µL 576 µL 1.15 mL

1 M Spermidine 0.9 µL 7.2 µL 14.4 µL

Cell Permeabilization Buffer - store at 4ºC for use on Day 2

Wash Buffer 1.4 mL 11.2 mL 22.4 mL

5% Digitonin 2.8 µL 22.4 µL 44.8 µL

Antibody Buffer - store on ice for use on Day 1

Cell Permeabilization Buffer 100 µL 800 µL 1.6 mL

0.5 M EDTA 0.4 µL 3.2 µL 6.4 µL

SECTION II: BEAD ACTIVATION (~30 MIN)

1. Gently resuspend the ConA Beads by pipetting. Transfer 11 μL/sample to a 1.5 mL tube for batch processing.

* NOTE: Batch processing at this step is recommended to improve sample handling. If a 1.5 mL tube magnet is not available, the beads can be processed individually (10 μL/sample) in the provided 8-strip PCR tubes using a compatible 8-strip magnet.

2. Place the tube on a magnet until slurry clears and pipette to remove sup.

* IMPORTANT: For all steps involving magnetic racks, take care to avoid disturbing the immobilized beads with pipette tips.

3. To avoid drying the beads, immediately add 100 μL/sample cold Bead Activation Buffer. Pipette gently to mix.

4. Place the tube on a magnet until slurry clears and pipette to remove sup. Repeat previous step for total of two washes.

5. Resuspend beads in 11 μL/sample cold Bead Activation Buffer.

*NOTE: If not batch processing, use 10 μL/sample at this step. Proceed directly to Section III.

6. For each experimental condition, aliquot 10 μL/sample of activated bead slurry into separate 8-strip tubes. Keep on ice until needed.

SECTION III: BINDING CELLS TO ACTIVATED BEADS (~30 MIN)

* NOTE 1: For sample inputs other than native, non-adherent cells (e.g. adherent cells, cryopreserved cells, tissue, nuclei, and cross-linked cells) see Appendix II.

* NOTE 2: Determine cell or nuclei integrity before and after conjugation to ConA beads. See Quality Control Checks: Cell/Nuclei Integrity and Bead Conjugation Check.

* IMPORTANT: Beads are prone to clumping; frequently vortex/pipette to even supension.

7. Harvest 0.5 million cells/sample in 1.5 mL tube. Centrifuge at 600 x g, 3 min at RT. Decant or pipette culture media sup.

8. Resuspend cells in 100 μL/sample RT Wash Buffer. Pipette to thoroughly resuspend. Centrifuge at 600 x g, 3 min, RT. Decant or pipette sup.

9. Repeat previous step for total of two washes.

10. Resuspend cells in 105 μL/sample in RT Wash Buffer and thoroughly pipette to mix. Aliquot 100 μL washed cells to each 8-strip tube containing 10 μL of activated beads. Gently vortex and/or pipette until evenly resuspended.

15

11. Incubate cell-bead slurry on benchtop for 10 min at RT to adsorb cells to beads.

SECTION IV: ANTIBODY BINDING (~30 MIN + OVERNIGHT)

12. If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice.

* IMPORTANT: If processing > 8 samples (multiple 8-strip tubes), remove & replace sups for a single strip before processing the next to avoid bead dry out during wash steps.

* NOTE: Multi-channel pipetting is highly recommended through the rest of the experiment. This helps to avoid bead dry out, improves yield, and increases experimental throughput.

13. Place the 8-strip tubes on an 8-strip tube magnet (high volume setting) until slurry clears. Pipette to remove sup, taking care to avoid disturbing the immobilized beads with pipette tip.

14. Immediately add 50 μL cold Antibody Buffer to each sample and gently vortex and/or pipette mix to prevent beads from drying.

15. Add 2 µL CUTANA H3K4 MetStat Spike-in Control dNucs to the cell samples designated for the positive (H3K4me3) and negative (IgG) control antibodies.

* IMPORTANT: The CUTANA spike-ins provide an essential defined control to ensure assay conditions are optimized (see Quality Control Checks). If desired, leftover spike-in controls can also be added to any experimental samples using an H3K4 methylation antibody (H3K4me1, H3K4me2 or H3K4me3 targets).

16. Add 0.5 μg antibody to each sample and gently vortex.

* IMPORTANT: Be sure to include the H3K4me3 positive and Rabbit IgG negative control antibodies in every experiment to monitor critical quality controls (see Quality Control Checks section). Control antibodies are at 0.5 mg/mL, therefore add 1.0 µL per sample.

* NOTE: Some antibodies are stored in glycerol solution and may be viscous. Take care to ensure accurate pipetting (e.g. aspirate slowly, check tip for accuracy, pipette up and down ~three times into CUT&RUN samples to clear any reamining glycerol from tip).

17. Incubate 8-strip tubes on nutator overnight at 4ºC while keeping capped ends slightly elevated to prevent beads from drying out.

18. Store the Cell Permeabilization Buffer at 4°C overnight for use on Day 2.

Experimental Protocol: Day 2

SECTION IV: ANTIBODY BINDING, CONTINUED (~10 MIN)

19. If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice. Fill with Cell Permeabilization Buffer.

* IMPORTANT: If processing > 8 samples (more than 1 x 8-strip tube), for subsequent wash steps remove & replace sups for a single strip before processing the next strip. This avoids bead dry out during wash steps.

20. Place the 8-strip tubes on magnet until slurry clears. Pipette to remove sup.

21. *While beads are on magnet*, add 200 μL cold Cell Permeabilization Buffer directly onto beads. Pipette to remove sup.

22. Repeat previous step for total of two washes, without removing 8-strip tubes from the magnet.

23. Add 50 μL cold Cell Permeabilization Buffer to each sample. Gently vortex and/or disperse clumps by thorough pipetting.

SECTION V: BINDING OF PAG-MNASE (~30 MIN)

24. Add 2.5 μL pAG-MNase (20x stock) to each sample. Gently vortex/pipette mix.

* NOTE: To evenly distribute enzyme across cells/nuclei, ensure beads are thoroughly resuspended by gentle pipetting with a P200.

25. Incubate samples for 10 min at RT. Return 8-strip tube to magnet. Remove sup.

26. *While beads are on magnet*, add 200 μL cold Cell Permeabilization Buffer directly onto beads. Pipette to remove sup.

27. Repeat previous step for total of two washes without removing 8-strip tubes from the magnet.

28. Remove 8-strip tubes from the magnet. Add 50 μL cold Cell Permeabilization Buffer to each sample. Gently vortex and disperse clumps by pipetting.

SECTION VI: TARGETED CHROMATIN DIGESTION AND RELEASE (~3 HRS)

29. Place 8-strip tubes on ice. Add 1 μL 100 mM Calcium Chloride to each sample and gently vortex. Ensure efficient digestion by making sure beads are thoroughly resuspended. Gently pipette with a P200 if needed.

17

30. Incubate 8-strip tubes on nutator for 2 hours at 4ºC.

31. Add 33 μL Stop Buffer to each sample. Gently vortex to mix.

32. Prior to first use, reconstitute E. coli Spike-in DNA in 200 μL DNase free water.

* IMPORTANT: Lyophilized DNA pellet is invisible to the eye. Prior to opening, pellet DNA by quick spin in a benchtop microfuge. After reconstitution, vortex tube on all sides to ensure complete resuspension.

33. Add 1 μL (0.5 ng) Spike-in DNA to each sample. Gently vortex to mix.

* NOTE: In general, aim for Spike-in DNA to comprise 0.5 – 5% (ideally closest to 1%) of total read counts in the sequencing data. Therefore, while 0.5 ng is a good starting amount for both high (e.g. H3K27me3) and low (e.g. H3K4me3) abundance targets, this may need to be adjusted higher or lower depending on the antibody used, target of interest, total DNA yield, and sequencing results.

34. Incubate 8-strip tubes for 10 min at 37ºC in a thermocycler.

35. Quick spin in benchtop microfuge.

36. Place 8-strip tubes on a magnet stand until slurry clears. Transfer sups containing CUT&RUN enriched DNA to 1.5 mL tubes and discard ConA Beads.

SECTION VII: DNA PURIFICATION (~30 MIN)

* NOTE: The DNA Cleanup Columns will retain fragments > 50 bp. For specific recommendations regarding smaller fragment size enrichment for TF binding studies, see FAQs, Appendix III.

37. Add 420 μL DNA Binding Buffer to each sample. Mix well by vortexing.

38. For every sample, place a DNA Cleanup Column into a DNA Collection Tube. Load each sample onto a column and label the top.

39. Centrifuge at 16,000 x g, 30 sec, RT. Discard the flow-through. Place the column back into the collection tube.

* NOTE: A vacuum manifold can be used in place of centrifugation. For each step, add the indicated buffer, turn the vacuum on, and allow the solution to pass through the column before turning the vacuum off.

40. Prior to first use, add 20 mL ≥ 95% ethanol to DNA Wash Buffer.

SECTION VII: DNA PURIFICATION (~30 MIN), CONTINUED

41. Add 200 μL DNA Wash Buffer to each sample column.

42. Centrifuge at 16,000 x g, 30 sec, RT. Discard the flow-through. Place the column back into the collection tube.

43. Repeat for a total of two washes.

44. Discard the flow-through. Centrifuge one additional time at 16,000 x g, 30 sec to completely dry the column.

45. Carefully remove the column from the collection tube, ensuring it does not come into contact with the flow-through. Transfer column to a clean pre-labeled 1.5 mL microfuge tube.

46. Elute DNA by adding 12 μL DNA Elution Buffer, taking care to ensure the buffer is added to the center of the column rather than the wall. Tap the column + microfuge collection tube on the benchtop to ensure all droplets are absorbed onto the resin.

* NOTE: 12 μL is recommended, however DNA can be eluted in 6 – 20 μL volumes depending on anticipated yield and desired final concentration. Larger elution volumes, longer incubation times, and/or multiple rounds of elution may improve DNA yield. However, sample concentration will be reduced with larger total elution volume.

47. Let sit 5 minutes, then centrifuge at 16,000 x g, 1 min, RT.

48. Vortex eluted material and use 1 μL to quantify the CUT&RUN-enriched DNA using the Qubit™ fluorometer as per the manufacturer’s instructions. See Quality Control Checks section for typical DNA yields.

49. CUT&RUN DNA can be stored at -20ºC for future processing.

* IMPORTANT:In ChIP protocols, it is common practice to analyze isolated ChIP DNA using agarose gel or capillary electrophoresis to determine fragment size distribution prior to library preparation. However, electrophoretic analysis of CUT&RUN DNA prior to library preparation is NOT recommended, since the amount of DNA is likely to be below the sensitivity of detection for these approaches. Instead, proceed directly to library preparation.

19

Experimental Protocol: Library Preparation and NGS

SECTION VIII: NGS LIBRARY PREPARATION (~4 HRS)

50. Using 5-10 ng purified CUT&RUN-enriched DNA, prepare sequencing libraries using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (or equivalent). No fragmentation step is needed; start directly with end repair.

* NOTE: Although 5-10 ng is recommended, quality CUT&RUN libraries can be prepared when the amount of DNA is undetectable by Qubit™.

51. Follow the manufacturer’s instructions for the following steps:

a. End repair (no fragmentation step needed)

b. Universal adapter ligation

c. DNA cleanup using 1.0X AMPure® beads to sample volume (this will retain fragments > 150 bp)

* NOTE: The AMPure ratio should be optimized for user-specific sequencing conditions. For example, a reduced ratio of 0.8X beads:sample may reduce adapter dimer contamination (~125 bp peak).

d. PCR and primer indexing according to the following cycling parameters:

i. 45 sec @ 98ºC

ii. 15 sec @ 98ºC

iii. 10 sec @ 60ºC

iv. Repeat steps ii - iii for a total of 14X

v. 1 min @ 72ºC

* NOTE: The hybrid anneal/extension PCR cycling parameters are intended to enrich for ~100 – 700 bp DNA fragments which are in the sequencing range. Contaminating higher molecular weight species will not be PCR-enriched.

52. Perform DNA cleanup using a ratio of 1.0X AMPure® beads to sample volume (e.g. 50 μL AMPure® to 50 μL PCR product).

53. Elute DNA in 17 μL 0.1X TE buffer and use 1 μL to quantify the purified PCR product using the Qubit™ fluorometer as per the manufacturer’s instructions.

* NOTE: Typical yield for a purified, PCR amplified sequencing library is ~500 – 750 ng (30 – 50 ng/μL in a final recovered volume of 15 μL TE Buffer)

54. CUT&RUN libraries can be stored at -20ºC for future processing.

SECTION IX: ANALYSIS OF LIBRARY FRAGMENT SIZE (~1 HR)

55. Per sample, prepare 5 μL at 10 ng/μL for loading on the Agilent Bioanalyzer®.

* NOTE: Record the dilution factor in order to calculate the original sample molarity from the Bioanalyzer reported concentration (reported in nM for the desired DNA size range between 100 – 1,000 bp).

56. Load 1 μL of 10 ng/μL sequencing library on Agilent High Sensitivity DNA Chip as per manufacturer’s instructions.

* NOTE: Typical concentration for 15 μL final sequencing library (100 – 1,000 bp region) is 100 – 200 nM.

57. The final Bioanalyzer® trace should show predominant enrichment of mononucleosomes yielded by the H3K4me3 Positive Control Antibody (~150 bp + sequence adapter length, Figure 6).

FIGURE 6

Typical CUT&RUN Agilent Bioanalyzer®tracesfrom5ngPCRamplifiedlibrariespreparedusing positive (H3K4me3) and negative (IgG) control antibodies as well as an H3K27me3 antibody (EpiCypher #13-0030). H3K4me3 and H3K27me3 libraries are predominantly mononucleosomes as indicated by the peaks at 275 bp (~150 bp mononucleosome + 125 bp sequence adapters).

Fig 6

IgG

H3K4me3

H3K27me3

[bp]1038050030015035

[bp]1038050030015035

[bp]1038040020035

[FU]

300200100

0

[FU]

500

0

[FU]

500

0

21

SECTION X: ILLUMINA SEQUENCING

58. Based on Agilent Bioanalyzer® molarity calculations, pool libraries at the desired ratios.

* IMPORTANT: Paired-end sequencing is recommended to enable target footprinting by identifying both ends of MNase cleavage. A minimum of 50 nucleotide paired-end reads are required to read the barcoded DNA on the CUTANA Spike-in Control nucleosomes.

* IMPORTANT: In contrast to ChIP-seq, only 3-8 million paired-end reads are needed for adequate CUT&RUN coverage. For low abundance targets (e.g. H3K4me3), aim for 3-5 million reads per sample. For high abundance targets (e.g. H3K27me3), aim for 5-8 million reads per sample.

59. To obtain 3-5M PE reads for 8 samples, load 0.8 pM (500 μL) into a MiniSeq™ High Output Reagent Kit, 150-cycles (Illumina #FC- 420-1002).

* NOTE: Flow cells using the MiniSeq™ High Output Kit typically yield 25 – 40M clusters passing filter (25 - 40M PE reads).

60. To obtain 3-5M PE reads for > 48-80 samples, load 0.8 pM (1,500 μL) into a NextSeq™ 500/550 High Output Kit v2.5, 150-cycles (Illumina #20024907).

* NOTE: Flow cells using the NextSeq™ High Output Kit v2.5 typically yield 400M clusters passing filter (400M PE reads).

SECTION XI: DATA ANALYSIS

61. Align paired-end reads to the appropriate reference genome corresponding to the species used in the experimental samples (e.g. using Bowtie 2).

62. Count the occurrences of the barcoded DNA sequences in the CUTANA H3K4 MetStat Spike-in Control Panel using both R1 and R2 paired end reads. A shell script for CUTANA spike-in control alignment is available on the CUT&RUN Kit product page at www.epicypher.com.

63. To determine experimental success, first evaluate performance of the positive and negative (IgG) control samples (see Quality Control Checks section).

64. Proceed to further data analysis for experimental samples once the positive controls are confirmed to have produced the expected results. For experimental normalization using the E. coli Spike-in DNA, see Appendix I.

Quality Control Checks

* IMPORTANT: EpiCypher recommends including positive and negative control antibodies

and corresponding CUTANA Spike-in Control dNucs in every experiment. If the quality

control checks for the controls perform as expected, then proceeding to sequencing with all

experimental samples is recommended. If sequencing results for controls match the expected

profile, but the data for experimental targets are not satisfactory, further optimization

may be necessary (e.g. cell number, sample preparation, digitonin permeabilization,

antibody concentration, alternative antibody vendors, etc.). EpiCypher Technical Support

(techsupport@epicypher.com) will ask for data from the control antibodies and spike-in

control dNucs to assist troubleshooting.

RECOMMENDED EXPERIMENTAL DESIGN:

• Always include H3K4me3 positive control and IgG negative control antibodies alongside experimental samples.

• Add immobilized CUTANA H3K4 MetStat Spike-in Control dNucs into the samples designated for the H3K4me3 positive control and IgG negative control antibodies. This enables a quantitative measure of how much target (H3K4me3) is recovered relative to off-target modifications (H3K4me0, H3K4me1, H3K4me2), providing essential insight for troubleshooting problematic experiments.

• Start with 500,000 cells and optimize conditions before reducing cell number.

• K562 cells can be used as a control cell line since they are known to be permeabilized under standard digitonin concentration and reference data is available for comparison.

• For experimental cells, optimize permeabilization conditions by varying the amount of Digitonin. See protocol (next page) and example results (Figure 7).

• Ensure starting cells / nuclei are of sufficient quality for CUT&RUN and confirm binding to ConA beads. See protocol (p. 24) and example results (Figure 8).

• Antibodies that work well in ChIP-seq do not guarantee success in CUT&RUN. For targets of interest, try EpiCypher's CUT&RUN validated antibodies (https://www.epicypher.com/cut-and-run-compatible-antibodies), inquire at techsupport@epicypher.com for antibody recommendations, or source multiple antibodies to the same target in order to test performance in CUT&RUN. Note: antibody performance from other vendors can vary dramatically lot-to-lot.

23

FIGURE 7

Example cell permeabilization optimization experiment. The minimum amount of digitonin ("Dig")needed to permeabilize K562 cells was determined by varying the amount of 5% Digitonin added to thefinalCell Permeabilization Buffer. Trypan blue staining was used to evaluate permeability with an automated cell counter (live/dead cell viability analysis). Green cells (Trypan negative) are intact, whereas permeabilized/dead cells (Trypan positive) are red. Values (bottom right of each panel) indicate percent of dead/permeabilized cells. 0.01% digitonin is the minimum concentration necessary to permeabilize > 95% of total cells.

OPTIMIZATION OF CELL PERMEABILIZATION

1. Prepare Cell Permeabilization Buffers with varying [Digitonin] (e.g. Figure 7).

2. Harvest cultured cells needed for CUT&RUN (e.g. 5 - 500k cells / sample).

3. Incubate cells with the various Cell Permeabilization Buffers for 10 minutes.

4. Mix 10 µL cells + 10 µL 0.4% Trypan blue. Load 10 µL onto hemacytometer slide.

5. Count live (intact, Trypan -) vs. dead (permeabilized, Trypan +) cells and select the minimum concentration of Digitonin needed to permeabilize >95% of cells.

CELL/NUCLEI INTEGRITY AND BEAD CONJUGATION CHECK

* NOTE: 5 µL excess cell or nuclei resuspension volume per sample is included in the protocol to account for pipetting error. However, it may be prudent to prepare one extra sample for evaluation of sample integrity.

1. Prior to starting with ConA Bead conjugation (Experimental Protocol, Step 10), take 10 µL from washed cells or prepared nuclei and evaluate sample integrity as follows:

a. Add 10 µL of 0.4% Trypan blue.

b. Mix 10x times by pipetting.

c. Transfer 10 µL to a counting slide.

d. View under brightfield/phase microscope or cell counter.

e. Confirm sample integrity or troubleshoot as needed (see Table, p. 25).

2. Proceed with bead binding by adding 100 µL of washed cells or nuclei to 10 µL of activated ConA Beads.

3. Gently vortex and/or pipette to mix cell/nuclei : bead mixture [sample slurry] until thoroughly resuspended.

4. Incubate sample slurry on benchtop for 10 minutes. Cells/nuclei will bind to the ConA Beads.

5. Place tube(s) on magnet; transfer 10 µL from the sup [unbound fraction] to a fresh 1.5 mL tube; set aside for later use. Remove the remaining unbound fraction.

6. To prevent beads from drying, immediately add 50 µL cold Antibody Buffer (contains Digitonin to permeabilize cells) to immobilized beads and gently vortex/pipette mix sample slurry to an even resuspension.

7. Remove 10 µL from sample slurry and place in 1.5 mL tube. Place remaining sample slurry on ice.

8. To 10 µL samples that were set aside (unbound fraction, sample slurry); perform Trypan staining as described above (Step 1a – e).

9. Continue on with the Experimental Protocol, Step 15: addition of CUTANA H3K4 MetStat Spike-in Control dNucs and antibodies.

25

FIGURE 8

Representative images of cell and nuclei samples for ConA Bead conjugation. Samples were stainedwithTrypanblueandvisualizedunderbrightfieldmicroscope.(A)CellsbeforeConABeadconjugation. A dead cell (blue, Trypan positive) is circled in red, whereas the remaining live cells are bright white and round. (B) Nuclei before ConA Bead conjugation. An intact cell (bright white, Trypan negative) is circled in red, whereas isolated nuclei are Trypan stained (see Appendix II; Sample Preparation: Nuclei). (C) Unbound fraction shows little to no material leftover after ConA Bead conjugation. (D) Representative sample slurry image showing nuclei (blue) successfully conjugated to activated ConA Beads (brown specks).

Sample Success Metrics (Tryptan Blue Stained)

Troubleshooting Tips

CellsFigure 8A

Cells should be bright white (Trypan excluded), round, and show >95% viability.

Evaluate cell culture conditions; use fresh media, determine

contamination issues.

NucleiFigure 8B

Nuclei should be >95% “dead” (Trypan blue

positive) and unclumped.

See Appendix II; Sample Preparation: Nuclei for a

detailed nuclei prep protocol.

Unbound FractionFigure 8C

Little to no material should be present if

conjugation occurred.Ensure that ConA Beads were

never frozen, cells/nuclei were not clumped, beads did not become

clumped or dried out, and all buffers were correctly prepared.

Sample SlurryFigure 8D

Successful conjugation in Antibody Buffer will show permeabilized cells/nuclei

surrounded by beads.

BEFORE DECISION TO SEQUENCE:

• The yield of CUT&RUN enriched DNA can vary in different experimental settings, starting cell numbers, and for different antibodies (even for antibodies to the same target). Therefore, DNA yield should not be used as a definitive metric of experimental success. However, in general the yield from the H3K4me3 positive control antibody should be slightly higher than IgG negative control. Typical results generated using 500,000 K562 cells are shown:

* NOTE: Total yields are influenced by a number of considerations, including starting number and type of cells, experimental perturbations, antibody specificity, antibody efficiency, and epitope abundance in each cell type. Yields shown are from 500,000 native K562 cells and are prior to PCR amplification.

• Yield of library prep and PCR (~750 ng from 5-10 ng library input, see Section VIII).

• Fragment size distribution analysis after library preparation (e.g. Agilent Bioanalyzer® or Tapestation® traces) should show enrichment of mononucleosomal fragments (~275 bp = ~150 bp nucleosome + 125 bp adapters, Figure 6).

* NOTE: Although it may be tempting to analyze fragment size distribution prior to library preparation, remember that CUT&RUN enriched DNA is often below the limit of detection for such approaches. Proceed directly to library preparation after CUT&RUN.

Target Target Abundance in Cells

Antibody ID Typical Yield* (ng DNA)

IgG (Negative Control)

None EpiCypher #13-0042

~2-5

H3K4me3 (Positive Control)

Low EpiCypher #13-0041

~5-10

H3K27me3 High Thermo Fisher #MA5-11198

~20-50

27

QUALITY CONTROL CHECKS AFTER SEQUENCING:

• Ensure appropriate read depth (3-8 million reads per sample) was achieved.

• Majority of reads (> 80%) should align uniquely to the species genome.

• Sequence duplication levels should be low (< 10% of total reads), but can be filtered out if needed.

• CUTANA Spike-in Control dNucs produce the expected results (see next section).

• E. coli Spike-in DNA comprises ~1% of total sequence reads (see Appendix I).

• Positive (H3K4me3) and negative (IgG) antibody controls produce the expected distribution and peak structure in the species tested. Results for experimental replicates should be highly reproducible. Representative examples for human K562 cells are shown (Figures 9 and 10).

FIGURE 9

Genome-wide analysis of CUT&RUN data presented as a heatmap of signal intensity for Rabbit IgG Negative Control Antibody, H3K4me3 Positive Control Antibody, and H3K27me3 antibody (EpiCypher #13-0030). High (red) and low (blue) signal aligned to the TSS (+/- 2 kb) of 18,793 genes are ranked by H3K4me3 intensity (top to bottom). Gene rows are aligned across the conditions. The heatmaps show that H3K4me3, a mark of active gene transcription, is enriched proximal to the TSS and is anti-correlated with H3K27me3, a mark of transcriptional repression. IgG shows low nonspecificbackgroundsignal.

Fig 8

IgG H3K4me3 H3K27me3

18,7

93 g

enes

alig

ned

to T

SS

TSS-2kb +2kb0

FIGURE 10

Data generated by four independent experimenters are virtually indistinguishable, demonstrating the reproducibility of the CUTANA ChIC/CUT&RUN Kit. Representative genome browser tracks showing CUT&RUN data generated by four independent experimenters using 500,000 K562 cells with Rabbit IgG Negative Control Antibody, H3K4me3 Positive Control Antibody, and H3K27me3 antibody (EpiCypher #13-0030). Total read counts were ~3 million for IgG and H3K4me3 samples, and ~6 million for H3K27me3. H3K4me3 tracks show sharp peaks localized to the TSS, while H3K27me3 tracks show broad peaks spread over transcriptionally repressed genomic regions. IgG shows lownonspecific background enrichmentwhen scaled to the positive controlH3K4me3 track.

Fig 9

LOC284865 RTN4R

H3K4me3

H3K27me3

IgG

DGCR6LDGCR8 TRMT2A ZDHHC8

[0-216]

[0-29]

[0-100]

[0-140]

[0-1481]

[0-547]

[0-250]

[0-965]

[0-500]

[0-500]

[0-500]

29

CUTANA SPIKE-IN CONTROLS

The CUT&RUN Kit includes a spike-in control panel consisting of four DNA-barcoded designer nucleosomes (H3K4me0, H3K4me1, H3K4me2 and H3K4me3) that are to be individually conjugated to streptavidin magnetic beads through biotinylated linker DNA (250x601, Figure 11). Just prior to antibody addition, these beads are spiked into CUT&RUN positive (H3K4me3) and negative (IgG) antibody controls alongside ConA-immobilized cells. pAG-MNase cleavage of the H3K4me3 nucleosome linker DNA in the anti-H3K4me3 positive control sample should specifically release this standard into solution. After sequencing, the relative read count of each spike-in nucleosome provides a quantitative metric of on- vs. off-target PTM recovery (Figure 12), thereby gauging experimental success and guiding troubleshooting efforts for problematic experiments.

Fig 10

250 bp construct

80 bp linker 123 bp Widom 601 sequence 22 bpbarcode

25 bplinkerBiotin

Positioned Nucleosome

H3K4me0

SA Bead

H3K4me1

SA Bead

H3K4me2

SA Bead

H3K4me3

SA Bead

FIGURE 11

Schematic of CUTANA H3K4 MetStat Spike-in Control dNucs, depicting the barcoded designer nucleosomes individually conjugated to magnetic streptavidin beads through biotinylated linker DNA.

Expected results using the CUTANA Spike-in Control dNucs (Figure 12):

• Overall read counts for the spike-in panel comprise ~1-8% of total sequencing reads.

• H3K4me3 Positive Control Antibody (EpiCypher 13-0041) shows strong enrichment for H3K4me3 spike-in nucleosomes with less than 5% cross-reactivity to off-target PTMs in the panel, but can be higher in cross-linked samples (~5-15%).

• IgG Negative Control Antibody shows no clear preference for a particular nucleosome in the panel.

• For results that deviate from these conditions: see suggested troubleshooting approaches, next page.

Antibody (Target, Vendor, Cat #)

IgG H3K4me1 H3K4me2 H3K4me3 H3K4me3EpiCypher EpiCypher EpiCypher EpiCypher Shah et al.13-0042 13-0040 13-0027 13-0041 abMe3-1

H3K4me0 33 2 2 1 5

H3K4me1 23 100 4 2 9

H3K4me2 21 6 100 2 122

H3K4me3 23 2 2 100 100

% on target 23% 91% 93% 96% 42%

% total reads 1.2% 3.9% 0.8% 2.1% 2.2%CU

TAN

A S

pike

-in

dNuc

FIGURE 12

CUTANA H3K4 MetStat Spike-in Control dNucs were added to CUT&RUN samples alongside the indicated H3K4 methyl state antibodies (top). Sequencing reads were searched for instances of each barcoded nucleosome (left axis) and counts normalized to the on-target PTM (100%, blue). EpiCypher SNAP-ChIP® CertifiedAntibodies toH3K4me1,H3K4me2, andH3K4me3 recoveredtheirrespectivePTMtargetswithhighspecificity.AnH3K4me3antibodypreviouslyfoundtocross-react with H3K4me2 in ChIP (abMe3-1, Shah et al.4) has similar cross-reactivity in CUT&RUN. The percentage of reads aligned to the on-target dNuc (relative to the total spike-in reads; % on target) and spike-in read counts (relative to total sequencing reads; % total reads) are shown (bottom).

31

Problem Troubleshooting Approach

Using anti-H3K4me3 & IgG Control Antibodies:CUTANA Spike-in Controls produce expected result (Figure 12), but genomic signal is poor (e.g. low signal-to-noise, poor peak structure).

• pAG-MNase cleavage and wash conditions are optimized.

• Confirm cell viability: low viability will increase noise.• Start with 500k cells & optimize controls before

decreasing cell number.• Ideally, start with native cells. If using cross-linked

samples, make sure they are only lightly cross-linked (see Appendix II).

• Ensure cells are not clumped prior to immobilization and beads do not clump throughout protocol.

• Ensure cells are adequately permeabilized by optimizing the amount of digitonin (see Figure 7).

• Optimize conditions in native K562 cells before trying additional sample types.

Using anti-H3K4me3 & IgG Control Antibodies:Both CUTANA Spike-in Controls and samples yield poor/no data.

• Indicates a fundamental failure in the workflow that impaired antibody binding, pAG-MNase cleavage, or adequate DNA recovery.

• Always start with 500k cells & optimize controls before decreasing cell number, particularly if this problem is accompanied by low DNA yield.

• Ensure Wash, Cell Permeabilization, and Antibody Buffers are prepared fresh the day of use.

• Carefully re-read the protocol, taking care to pay atten-tion to important notes.

• Ensure beads do not clump or stick to side of tube throughout protocol. Gently pipette and vortex to mix as needed. Ensure digitonin is included in both the Cell Permeabilization and Antibody Buffers.

H3K4me3 & IgG Control Antibodies produce the ex-pected results for CUTANA Spike-in Controls (Figure 12) and genomic signal (Figures 9-10). However, antibodies to additional targets show no clear enrichment.

• The workflow is optimized for controls.

• Explore alternate antibodies to the target. Using a “ChIP-grade” antibody does not guarantee success in CUT&RUN. EpiCypher has conducted extensive testing to a variety of histone and non-histone targets in ChIP and CUTANA assays. Contact us for recommendations.

• Some targets may benefit from light cross-linking, although yield may be impacted. See Appendix II.

• Ensure the target of interest is expressed (or localized to chromatin) in the study cells/conditions used. Increase cell number or stimulate target expression if needed.

Appendix I: Experimental Normalization Using E. coli Spike-in DNA

1. Aim for E. coli Spike-in DNA to comprise ~1% (0.5-5%) of total sequencing reads. In the protocol, 0.5 ng is recommended to start. Based on target abundance, antibody efficiency and experimental conditions, the spike-in amount may need to be adjusted higher or lower to achieve read counts in this range.

2. After sequencing, in addition to the experimental sample reference genome (e.g. human hg19 build), align reads to the E. coli K12, MG1655 reference genome. Filter out reads that do NOT align uniquely. https://support.illumina.com/sequencing/sequencing_software/igenome.html

3. For pairwise comparisons, quantify E. coli Spike-in DNA reads for treatment and untreated samples and normalize to sequencing depth.

For example:

Treatment spike-in = 100,000 Ec reads in 5,000,000 total reads = 2%

Untreated spike-in = 30,000 Ec reads in 3,000,000 total reads = 1%

4. Calculate normalization factor (see Tay et al.5) such that after normalization the E. coli spike-in signal is set to be equal across all samples.

For example:

Treatment normalization factor = 1 / 2% spike-in bandwidth = 0.5

Untreated normalization factor = 1 / 1% spike-in bandwidth = 1.0

5. Use single scalar normalization ratio with the --scaleFactor option enabled in deeptools bamCoverage tool (https://deeptools.readthedocs.io/en/develop/con-tent/tools/bamCoverage.html) to generate normalized bigwig files for visualization in IGV.

For example:

Treatment sample --scaleFactor = 0.5

Untreated sample --scaleFactor = 1.0

NOTE: The effect of normalization on a sample is inversely proportional to the E. coli spike-in bandwidth. In other words, samples with the highest bandwidth will receive the largest reduction in signal after normalization. For further information on experimental normalization using exogenous spike-in controls, see Tay et al. (2020)5 and Orlando et al. (2014)6.

33

Appendix II: Protocol Variations

* NOTE: The kit is compatible with fresh cells, as well as cryopreserved cells, cross-linked

cells, and isolated nuclei (Figure 3). The protocols below provide general guidelines and

recommendations for working with these sample preparations.

SAMPLE PREPARATION: ADHERENT CELLS

Enzymatic treatment (e.g. trypsin) will degrade glycoproteins, thereby impairing cell adsorption to the ConA Beads. Therefore, do not use trypsin to prepare adherent cells for CUT&RUN. Instead, we recommend scraping adherent cells to dislodge them from the dish, then pellet by centrifugation for 600 x g, ~3 min at RT. Proceed directly to the CUT&RUN Wash Buffer steps (Experimental Protocol: Section III).

SAMPLE PREPARATION: CRYOPRESERVATION

Cells should be freeze/thawed under conditions that minimize lysis, which can contribute to elevated background in CUT&RUN. Supplement cell culture media with a cryoprotective agent (e.g. 10% DMSO in media) and slow freeze samples (-1ºC per minute) in an isopropanol-filled container placed at -80ºC (e.g. “Mr. Frosty” or equivalent). When ready to perform CUT&RUN, quickly and completely thaw the samples at 37ºC and prepare cells as described (Experimental Protocol: Section III).

SAMPLE PREPARATION: TISSUES

The primary requirement for CUT&RUN is that tissue is processed into a mono- dispersion of cells, typically by mechanical maceration/douncing. EpiCypher has not currently investigated tissue-specific protocols, however a number of groups have reported methods for performing CUT&RUN using human and mouse primary tissues7-10.

SAMPLE PREPARATION: IMMUNE CELLS

Concanavalin A (ConA) is a lectin, which can cause immune cell activation through interaction with cell surface receptors. For studies involving immune cells, it is recommended to isolate nuclei. See detailed protocol for nuclei isolation, next page.

SAMPLE PREPARATION: NUCLEI

1. Prepare 200 μL / sample (+ extra dead volume) Nuclear Extraction Buffer fresh the day of use. Sterile Filter.

2. Add 1:2,000 dilution of 1 M Spermidine and 1X Protease Inhibitor to the Nuclear Extraction Buffer. Place final buffer on ice.

3. Harvest 0.5 million cells per sample (+ extra for Trypan blue staining) by centrifugation for 3 min at 600 x g, RT. Remove and discard supernatant (sup).

4. Resuspend cells in 100 μL per sample 1X PBS. Set aside 10 μL cells for future analysis by Trypan blue staining (intact cell control). Centrifuge at 600 x g, 3 min, RT. Remove and discard sup.

* NOTE: For all steps, the ratio of buffer volumes : cells scales linearly. For example, use 1 mL buffer for 5 x 106 cells.

5. Resuspend cells in 100 μL per sample cold Nuclear Extraction Buffer.

6. Incubate samples on ice for 10 min.

7. Centrifuge at 600 x g, 3 min, 4ºC. Remove and discard sup. The pellet should change in appearance from sticky, pale yellow (cells) to white and fluffy (nuclei).

Materials Needed Recipe / Source

Nuclear Extraction Buffer 20 mM HEPES, pH 7.9, 10 mM KCl,0.10% Triton X-100, 20% glycerol

Protease Inhibitor Roche #11873580001

1 M Spermidine EpiCypher CUTANAChIC / CUT&RUN Kit #21-1005

Phosphate Buffered Saline (PBS) Any vendor

0.4% Trypan blue Invitrogen #T10282

Brightfield or phase microscope +hemacytometer slides

Any vendor

35

8. Resuspend nuclei in 105 μL per sample cold Nuclear Extraction Buffer. Set aside 10 µL nuclei for Trypan blue staining.

9. Add 10 uL Trypan blue to the intact cell control (Step 4) and the isolated nuclei (previous step). Mix 10x times by pipetting.

10. Load onto hemacytometer slide and examine under brightfield/phase microscope to determine whether nuclei have been efficiently isolated (Figure 13).

11. To cryopreserve nuclei, slowly freeze samples in isopropanol-filled chiller in -80ºC freezer.

12. When ready to use samples for CUT&RUN, thaw nuclei quickly by placing on 37ºC block.

13. Proceed to CUT&RUN ConA Bead conjugation step (Experimental Protocol, Step 10).

FIGURE 13

Morphology characteristic of intact K562 cells (left) compared to isolated nuclei (right) when visualizedunderbrightfieldmicroscopeafterTrypanbluestaining.Isolatednucleiwillstainblue,whilecells will be bright white and round. For accurate nuclei counts, record "dead" cell numbers on an automated cell counter or manually count blue stained nuclei.

CELLS NUCLEI

SAMPLE PREPARATION: CROSS-LINKING

It is recommended to first try native samples when possible, as this works well for the majority of targets. However, CUT&RUN signal for labile targets or highly transient chromatin binding proteins may be improved by cross-linking.

In particular, histone deacetylase activity may contribute to incomplete low resolution genomic profiles in native CUT&RUN. In these cases, light (e.g. 0.1% formaldehyde, 1 min) to moderate (e.g. 1% formaldehyde, 1 min) cross-linking may improve signal even though total yield may be reduced. However, heavy cross-linking such as that typically used for ChIP-seq (e.g. 1% formaldehyde, 10 min) has been observed to damage histone acetylation signal in K562 cells (Figure 14). Therefore, optimal cross-linking conditions for profiling histone acetylation should be empirically determined in the model system of interest. The protocol described provides general guidelines for optimizing fixation conditions.

FIGURE 14

H3K27acCUT&RUNsignalisimprovedbylighttomoderatefixation(0.1–1%formaldehydefor1min)comparedtonativecells.However,heavy(1%,10min)cross-linkingsignificantlyreducesCUT&RUN DNA yield. CUT&RUN data from 500,000 K562 cells are displayed in a heatmap with each gene row aligned across the conditions.

Fig 11

Freshcells

(native)

Lightcross-link(0.1% 1m)

Moderatecross-link(1% 1m)

18,7

93 g

enes

alig

ned

to T

SS

TSS

Standardcross-link(1% 10m)

-2kb +2kb

37

1. From suspension tissue culture, transfer 0.5 million cells per sample into a 15 or 1.5 mL tube. For adherent cells, cross-link cells while still attached to plate.

2. Add 37% formaldehyde directly to culture to achieve desired final concentration of formaldehyde (recommended ~0.1% - 1%).

* NOTE: In initial experiments, test a range of formaldehyde concentrations to determine the optimal fixation conditions for the cell type and target of interest.

3. Quickly vortex suspension cells or swirl plate for adherent cells to mix. Incubate for 1 - 10 min at RT.

* NOTE: In initial experiments, test a range of times to determine optimal fixation conditions.

4. Quench fixation reaction by adding glycine to a final concentration of 125 mM. Vortex/swirl to mix. Scrape adherent cells from the plate, transfer to 15 mL tube, and centrifuge at 600 x g, 3 min, RT.

5. Begin CUT&RUN by starting at the Experimental Protocol, Step 7. Carry through the protocol with the following modifications:

a. Use Wash, Cell Permeabilization, and Antibody buffers (Figure 5) supplemented with Triton X-100 and SDS (see table above).

b. After 37ºC incubation in the Experimental Protocol, Step 34, place 8-strip tubes on magnet stand until slurry clears. Transfer sups containing CUT&RUN enriched DNA to new 8-strip tubes.

c. Reverse cross-links by adding 0.8 μL 10% SDS and 1 μL of 20 μg/μL Proteinase K to each sample. Mix by vortexing. Incubate overnight at 55ºC using a thermocycler.

d. After incubation, quick spin samples in benchtop microfuge. Resume protocol at Step 37 and proceed as normal.

Materials Needed Recipe / Source

Pre-Wash Buffer supplemented with detergent

Add 1% Triton X-100 + 0.05% SDS to Pre-Wash Buffer (EpiCypher CUTANA

ChIC / CUT&RUN Kit #21-1002)

37% Formaldehyde Sigma #252549

Glycine Sigma #50046

10% SDS Any vendor

20 μg/μL Proteinase K Ambion #AM2546

Appendix III: Frequently Asked Questions

1. What types of cell inputs are compatible with CUT&RUN?EpiCypher has confirmed that CUT&RUN is compatible with whole cells and nuclei derived from mammalian suspension, adherent cancer lines, and nuclei derived from mouse primary B-cells. EpiCypher has not yet directly tested other cell types, but a number of groups have successfully performed CUT&RUN on human and mouse primary tissue7-10, FACS sorted11 and immune cells12,13. Note that lectins (e.g. ConA) play a role in the innate immune system. Therefore, immune cell types may be inadvertently stimulated via binding to ConA Beads. To circumvent this potential problem in CUT&RUN, EpiCypher recommends using nuclei11 or a crosslinking strategy14.

2. Is CUT&RUN compatible with frozen or cross-linked cells?Yes. EpiCypher has confirmed that freeze/thawed cells (e.g. slow freeze in 10% DMSO/media, and then quick thaw at 37ºC) generate data of indistinguishable quality to fresh material. EpiCypher has also tested previously reported cross-linking conditions and recommended wash buffers14, and found that while yields may be lower than native cells, the resulting data tracks display similar quality. See Appendix II for details.

3. Does EpiCypher’s CUT&RUN protocol work on non-PTM targets?Yes. The current protocol has been used at EpiCypher to generate numerous non-PTM CUT&RUN data, including CTCF, BRD4, and SMARCA4 (BRG1). No protocol modifications were necessary to generate these data since the DNA Cleanup Columns recover > 50 bp fragment sizes.

However, for TFs in particular, which generate sub-nucleosomal size (< 120 bp) DNA fragments, modifications to the library protocol have been reported9,15 to improve the representation of these smaller fragments during library preparation. Briefly, to enrich for sub-120 bp fragments, the authors reduced the inactivation temperature after end repair from 65ºC for 30m to 50ºC for 1 hr. Also, after the ligation reaction, the AMPure® bead size-selection ratio was increased to 1.75X volume.

For TF CUT&RUN fragment sizes, both nucleosomal (~150 bp) and sub-nucleosomal (< 120 bp) reflect TF chromatin occupancy locations, however, the sub-nucleosomal fragments provide higher resolution mapping for their locations.

39

4. What antibodies does EpiCypher recommend for CUT&RUN?Antibodies that work well in ChIP may not always work in CUT&RUN. To address this unmet need for researchers, EpiCypher has developed “CUTANA Compatible Antibodies” (see the expanding list of targets at: https://www.epicypher.com/cut-and-run-compatible-antibodies). Every lot of a CUTANA Compatible Antibody is validated directly in CUT&RUN and/or CUT&Tag. For chromatin associated protein targets, the antibody is determined to yield peaks that show a genomic distribution pattern consistent with reported function(s) of the target protein (for example, DNA binding motif analysis for transcription factors). For histone PTM targets, antibodies are particularly susceptible to off-target cross reactivity which can compromise biological interpretations4. EpiCypher has conducted extensive studies of histone PTM antibodies in ChIP-seq by using defined spike-in controls in situ to show that the majority of antibodies are not fit-for-purpose4 and data in preparation

(also see: https://www.epicypher.com/blog/choosing-chip-antibodies). EpiCypher is currently developing full panels of recombinant nucleosome controls (widely studied histone lysine methylation and acylation states) that are uniquely designed for CUT&RUN, similar to the CUTANA H3K4 MetStat spike-in controls included in this kit. CUTANA spike-in control panels will be used for antibody validation similiar to EpiCypher's rigorously validated SNAP-ChIP Certified Antibodies (see: https://www.epicypher.com/technology/snap-chip-antibodies). For more information or for antibody recommendations, please contact techsupport@epicypher.com.

5. What pipeline is recommended for CUT&RUN sequencing analysis? Do the same bioinformatic methods that are used for ChIP-seq also work for CUT&RUN data? CUT&RUN data analysis methods have been described in detail1,3. In short, a very similar approach can be used compared to ChIP-seq with a few key considerations. Raw sequence reads can be aligned using Bowtie 216. The Integrative Genomics Viewer (IGV)17 and/or DeepTools18 can be used for data visualization (e.g. bigWig files graphed over a genome browser to visualize enrichment patterns). There are special considerations for peak calling since CUT&RUN has very low background compared to ChIP-seq and an “Input” sample is not sequenced. MACS219, a standard peak calling program used for ChIP-seq analysis, also works well in CUT&RUN. However SEACR20 , a peak caller specifically designed for CUT&RUN, has also been published. SEACR can be compared with MACS2 to determine which pipeline most faithfully represents the specific dataset of interest. For chromatin-associated proteins including transcription factor analysis, CUT&RUNTools21 is uniquely designed for CUT&RUN data processing including DNA footprinting analysis.

Appendix IV: Safety Datasheet

Component Name

Bead Activation BufferPre-Wash BufferStop BufferDNA Binding BufferDNA Wash BufferDNA Elution Buffer1 M Spermidine5% Digitonin0.5 M EDTA100 mM Calcium ChlorideConA BeadspAG-MNaseRabbit IgG Negative Control AntibodyH3K4me3 Positive Control AntibodyCUTANA K-MetStat Spike-in ControlsSA BeadsE. coli Spike-in DNA

Product Identification

Product Name: CUTANA ChIC/CUT&RUN KitSynonyms: None.Molecular Weight: Not applicable to mixtures.Chemical Formula: Not applicable to mixtures.Recommended Use: This product is for research and development only.

Hazardous Ingredients

NANANAYesNANANANANANANANANANANANANA

EpiCypher, Inc.

6 Davis Dr. Ste 755 Durham, NC 27709 USA

Phone: 1-855-374-2461 Fax: 1-855-420-6111 Email: info@epicypher.com

www.epicypher.com

24 Hour Emergency Phone Number:

US & Canada: 1-800-535-5053

International: 1-352-323-3500

41

Signal Word

WARNING

Hazard Statements

Harmful if swallowed, causes skin irritation, causes serious eye irritation, flammable liquid and vapor.

Precautionary Statements

Store in a well-ventilated place. Keep container tightly closed.

Prevention• Wear protective gloves/protective clothing/eye protection/face protection.

• Wash face, hands and any exposed skin thoroughly after handling.

• Do not eat, drink or smoke when using this product.

• Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.

• Ground/bond container and receiving equipment.

• Use only non-sparking tools.

• Take precautionary measures against static discharge.

Hazardous Identification

DNA Binding Buffer

Label Elements

Acute toxicity – Oral Category 4Skin corrosion/irritations - Category 2Serious eye damage/eye irritation - Category 2Flammable liquids - Category 3

Classification

This chemical is considered hazardous by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200)

Hazardous Identification continued

Chemical Name

Isopropyl AlcoholSodium PerchlorateGuanidine Hydrochloride

Kit Volume

< 11 ml< 2 ml< 8 ml

CAS Number

67-63-07601-89-050-01-1

Composition and Information on Ingredients

DNA Binding Buffer (mixture)

Precautionary Statements

Response

• IF EXPOSED OR CONCERNED: Get medical advice/attention.

• IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. If eye irritation persists: Get medical advice/attention. If skin irritation occurs: Get medical advice/attention.

• IF ON SKIN (OR HAIR): Take off immediately all contaminated clothing. Rinse skin with water/shower. Wash contaminated clothing before reuse.

• IF SWALLOWED: Call a POISON CENTER or doctor if you feel unwell.Rinse mouth.

• IN CASE OF FIRE: Use CO₂, dry chemical, or foam to extinguish.

First Aid Measures

General advice: If symptoms persist, call a physician. Do not breathe dust/fume/gas/mist/vapors/spray. Do not get in eyes, on skin, or on clothing.

Inhalation: Remove to fresh air. If breathing is irregular or stopped, administer artificial respiration. Avoid direct contact with skin. Use barrier to give mouth-to-mouth resuscitation. If symptoms persist, call a physician.

Eye contact: Rinse immediately with plenty of water, also under the eyelids, for at least 15 minutes. Keep eye wide open while rinsing. Do not rub affected area. Remove contact lenses, if present and easy to do. Continue rinsing. Get medical attention if irritation develops and persists.

43

First Aid Measures continued

Skin contact: Wash off immediately with soap and plenty of water while removing all contaminated clothes and shoes. Wash contaminated clothing before reuse. If skin irritation persists, call a physician. Immediate medical attention is not required.

Ingestion: Do NOT induce vomiting. Clean mouth with water and drink afterwards plenty of water. Never give anything by mouth to an unconscious person. If symptoms persist, call a physician.

Most important symptoms and effects: May cause redness and tearing of the eyes. Prolonged contact may cause redness and irritation. Vapors may cause drowsiness and dizziness.

Note to Physicians: Treat symptomatically.

Fire Fighting Measures

Suitable Extinguishing Media: Dry chemical. Carbon dioxide (CO₂). Water spray. Alcohol resistant foam.

Specific hazards arising from chemical: Risk of ignition. Keep product and empty container away from heat and sources of ignition. In the event of fire, cool tanks with water spray. Fire residues and contaminated fire extinguishing water must be disposed of in accordance with local regulations.

Special protective equipment for fire-fighters: In the event of a fire, wear full protective clothing and NIOSH-approved self-contained breathing apparatus with full facepiece operated in the pressure demand or other positive pressure mode.

Accidental Release Measures

Spill response: Absorb or cover with dry earth, sand or other non- combustible material and transfer to containers. Soak up condensate or spills with inert absorbent material and collect in ventilated waste container for disposal. Wear lab coat, chemical resistant gloves and safety glasses. Take precautionary measures against static discharges. Wash down the spill site.

Waste disposal method: Dispose of in accordance with all federal, state and local regulations.

Handling and Storage

Safe Handling: Use personal protection equipment. Avoid breathing vapors or mists. Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking. Use grounding and bonding connection when transferring this material to prevent static discharge, fire or explosion. Use with local exhaust ventilation. Use sparkproof tools and explosion-proof equipment. Keep in an area equipped with sprinklers. Avoid contact with skin, eyes or clothing. Take off contaminated clothing and wash before reuse.

Storage Conditions: Keep containers tightly closed in a dry, cool and well-ventilated place. Keep away from heat, sparks, flame and other sources of ignition. Keep in properly labeled containers. Do not store near combustible materials. Keep in an area equipped with sprinklers. Store in accordance with local regulations.

Exposure Controls / PPE

Exposure Limits: The following ingredients are the only ingredients of the product above the cut-off level which have an exposure limit applicable in the region for which this safety data sheet is intended or other recommended limit. At this time, the other relevant constituents have no known exposure limits from the sources listed here.

45

Exposure Controls / PPE continued

Isopropyl Alcohol

67-63-0

STEL: 400 ppm

TWA: 200 ppm

TWA: 400 ppm

TWA: 980 mg/m3 (vacated)

TWA: 400 ppm (vacated)

TWA: 980 mg/m3 (vacated)

STEL: 500 ppm (vacated)

STEL: 1225 mg/m3

IDLH: 2000 ppm

TWA: 400 ppm

TWA: 980 mg/m3

STEL: 500 ppm

STEL: 1225 mg/m3

Engineering Controls: Showers, eyewash stations, ventilation systems.Personal Protective EquipmentEye/face protection: Tight sealing safety goggles.Hand protection: Wear suitable impervious gloves.Skin and body protection: Wear suitable protective clothing, long sleeved clothing, Chemical resistant apron, and antistatic boots. Respiratory protection: no protective equipment is needed under normal use conditions. If exposure limits are exceeded or irritation is experienced, ventilation and evacuation may be required.

Appearance:Odor:Boiling Point:Melting Point:Solubility:Flash Point:Specific Gravity:pH:

Colorless LiquidAlcohol90 °C / 194 °FNo data availableNo data available27 °C / 80.6 °FNo data available5.10-5.30

Physical and Chemical Properties

Stability and Reactivity

Chemical Stability: Stable under ordinary conditions of use and storage.Hazardous Decomposition Products: None known based on information supplied.Incompatibilities: Strong oxidizers, strong acids and bases.Conditions to Avoid: Heat, flame, sparks, incompatibles.

Toxicological Information

Inhalation Specific test data for the substance or mixture is not available. May cause irritation of respiratory tract.Eye contact Specific test data for the substance or mixture is not available. Irritating to eyes. Causes serious eye irritation.Skin contact Specific test data for the substance or mixture is not available. Prolonged contact may cause redness and irritation. Repeated exposure may cause skin dryness or cracking.Ingestion Specific test data for the substance or mixture is not available. Ingestion may cause gastrointestinal irritation, nausea, vomiting and diarrhea. Harmful if swallowed. May cause drowsiness or dizziness.Symptoms related to the physical, chemical and toxicological characteristics Redness. May cause redness and tearing of the eyes.Acute toxicity Numerical measures of toxicity. The following values are calculated based on chapter 3.1 of the GHS document: ATEmix (oral) 1,127.90 mg/kg ATEmix (dermal) 4,599.60 mg/kg ATEmix (inhalation-dust/mist) 157.80 mg/Carcinogenicity Isopropyl Alcohol IARC Group 3Target organ effects Respiratory system, Eyes, Skin.

Ecological Information

Chemical Name Aquatic Plants Fish Crustacea

Isopropyl Alcohol 67-63-0

EC50: > 1000 mg/L (72h, Desmodesmus subspicatus)EC50: > 1000 mg/L (96h, Desmodesmus subspicatus)

LC50: =9640 mg/L (96h, Pimephales promelas) LC50: > 1400000 μg/L (96h, Lepomis macrochirus)LC50: =11130mg/L (96h, Pimepha les promelas.

EC50: =13299mg/L (48h, Daphnia magna)

Guanidine Hydrochloride 50-01-1

NA LC50: =1758mg/L (48h, Leucisc usidus)

NA

47

1. Skene & Henikoff. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6 (2017).

2. Schmid et al. ChIC and ChEC; genomic mapping of chromatin proteins. Mol. Cell 16, 147-157 (2004).

3. Skene et al. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat. Prot. 13, 1006-1019 (2018).

4. Shah et al. Examining the roles of H3K4 methylation states with systematically characterized antibodies. Mol. Cell 72, 162-177 (2018).

5. Tay et al. Hdac3 is an epigenetic inhibitor of the cytotoxicity program in CD8 T cells. J. Exp. Med. 217 (2020).

6. Orlando et al. Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep. 9, 1163-1170 (2014).

7. de Bock et al. HOXA9 cooperates with activated JAK/STAT signaling to drive leukemia development. Cancer Discov. 8, 616-631 (2018).

8. Janssens et al. Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs. Epigenetics Chromatin 11 (2018).

9. Liu et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 173, 430-442 (2018).

10. Uyehara & McKay. Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 116, 9893-9902 (2019).

11. Hainer et al. Profiling of pluripotency factors in single cells and early embryos. Cell 177, 1319-1329.e11 (2019).

12. Mathsyaraja et al. Max deletion destabilizes MYC protein and abrogates Emicro-Myc lymphomagenesis. Genes Dev. 33, 1252-1264 (2019).

13. Roth et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405-409 (2018).

14. Zheng & Gehring. Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN. Plant Reprod. 32, 63-75 (2019).

15. Zhu et al. CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis. Genome Biol. 20 (2019).

16. Langmead & Salzberg. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).

17. Robinson et al. Integrative Genomics Viewer. Nat. Biotechnol. 29, 24–26 (2011).

References

18. Ramírez et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 8, 44 (2016).

19. Gaspar. Improved peak-calling with MACS2. bioRxiv doi: 10.1101/496521(2018).

20. Meers et al. Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics Chromatin 12, 42 (2019).

21. Zhu et al. CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis. Genome Biol. 20, 192 (2019).

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24 Hour Emergency Phone Number:US & Canada: 1-800-535-5053

International: 1-352-323-3500

EpiCypher, Inc.6 Davis Dr. Ste 755Durham, NC 27709 USA

www.epicypher.comPh: 1-855-374-2461 F: 1-855-420-6111Email: info@epicypher.com Tech Support: techsupport@epicypher.com